Mutasyon Analiz Teknikleri I

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1 Mutasyon Analiz Teknikleri I
Hafta VII PCR Temelli Genetik Analiz Yaklaşımları Mutasyon Analiz Teknikleri I Doç. Dr. Hilâl Özdağ

2 DHPLC Transgenomic/WAVE

3 DHPLC http://insertion.stanford.edu/melt.html
BRCA ex1 CAATAGGTAGCGATTCTGACCTTCGTACAGCAATTACTGTGATGCAATAA GCCGCAACTGGAAGAGTAGAGGCTAGAGGGCAGGCACTTTATGGCAAACT CAGGTAGAATTCTTCCTCTTCCGTCTCTTTCCTTTTACGTCATCCGGGGG CAGACTGGGTGGCCAATCCAGAGCCCCGAGAGACGCTTGGCTCTTTCTGT CCCTCCCATCCTCTGATTGTACCTTGATTTCGTATTCTGAGAGGCTGCTG CTTAGCGGTAGCCCCTTGGTTTCCGTGGCAACGGAAAAGCGCGGGAATTA CAGATAAATTAAAACTGCGACTGCGCGGCGTGAGCTCGCTGAGACTTCCT GGACGGGGGACAGGCTGTGGGGTTTCTCAGATAACTGGGCCCCTGCGCTC AGGAGGCCTTCACCCTCTGCTCTGGGTAAAGGTAGTAGAGTCCCGGGAAA GGGACAGGGGGCCCAAGTGATGCTCTGGGGTACTGGCGTGGGAGAGTGGA TTTCCGAAGCTGACAGATGGGTATTCTTTGACGGGGGGTAGGGGCGGAAC CTGAGAGGCGTAAGGCGTTGTGAACCCTGGGGAGGGGGGCAGTTTGTAGG TCGCGAGGGAAGCGCTGAGGATCAGGAAGGGGGCACTGAGTGTCCGTGGG GGAATCCTCGTGATAGGAACTGGAATATGCCTTGAGGGGGACACTATGTC TTTAAAAACGTCGGCTGGTCATGAGGTCAGGAGTTCCAGACCAGCCTGAC CAACGTGGTGAAACTCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCG TGGTGCCGCTCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTAGAAC C with 851 base pairs Melting temperature of entire strand: 61.25° Temperature = 55° Celsius: 1-3, Temperature = 56° Celsius: 4-5, Temperature = 57° Celsius: , Temperature = 58° Celsius: 6-6, , , Temperature = 59° Celsius: 7-48, , , , Temperature = 60° Celsius: , , , , Temperature = 61° Celsius: , , , Temperature = 62° Celsius: , This sequence should be run at all of the following temperatures to ensure detection of all polymorphisms. Recommended temperature: 62° Recommended temperature: 56°

4 DHPLC CBP Exon 6 A. Rothan’dan

5 DHPLC A. Rothan’dan

6 DHPLC A. Rothan’dan

7 DHPLC Ezzeldin et al. Ann Biochem. 2002;306:63-73.

8 DHPLC

9 DHPLC DHPLC = denaturing high-performance liquid chromatography.
Ezzeldin et al. Ann Biochem. 2002;306:63-73.

10 Karıştırma testi DHPLC
P300/EXON16 4239G->T E1014X

11 Karıştırma testi DHPLC
CBP/EXON31.1 5371-8INSA

12 DHPLC HDAC2 EXON1 Karıştırma

13 HDAC2 143InsCAG

14 DHPLC HDAC2 EXON12 Karıştırma

15 HDAC2 1573DelA HCT15

16 HSUV39H2 -11InsA (?) SSCP

17 Kapiller Elektroforez

18 Genel Primerlerle işaretleme

19 P G>T (E1014X)

20 P G>T (E1014X )

21 CBP IVS21-4del22

22 CBP IVS21-4del22

23 CBP IVS31-7InsA

24 CBP IVS31-7InsA

25 TGCE Temperature Gradient Capillary Electrophoresis